The overall goal of our study is to develop an effective, dually active anthrax vaccine (DAAV) that confers simultaneous protection against both the bacilli and the toxins. Systemic anthrax infection by Bacillus anthracis results in extensive septicemia, toxemia, and almost invariably death. The pathogenesis of anthrax involves two crucial processes, i.e., replication of bacilli and release of toxins. The bacilli protect themselves with a poly-gamma-D-glutamic acid (PGA) capsule to evade immune surveillance while secreting large amounts of toxins. Hence, we hypothesize that an effective strategy needs to both kill the bacilli and destroy the toxins. We further hypothesize that a DAAV can be constructed based on the two major virulence factors, specifically, the bacillus capsular PGA and the toxin core protective antigen (PA). Our preliminary study demonstrates that PGA-PA conjugates induce potent anti-PA and anti-PGA antibodies and protect immunized mice against lethal challenges with both anthrax toxin and analogous bacilli. Such vaccines are superior to existing vaccines that solely target anthrax toxins. The focus of our proposed study is to further develop and optimize PA/PGA-based DAAVs. We will prepare libraries of three classes of DAAV constructs (DAAV-X, E, and C) by systematically varying their structures. We will derive optimal constructs by comparing their ability to elicit anti-PGA and anti-PA antibodies and their ability to protect mice against challenges with lethal toxins and virulent bacilli. Optimized DAAVs will be tested for their efficacy in protecting mice and rabbits against anthrax spore challenges. Aim 1. To optimize "cross-linked" PGA-PA conjugates (DAAV-X). Aim 2. To create and optimize "end-linked" PGA-PA conjugates (DAAV-E). Aim 3. To develop combination anthrax vaccines consisting of mixtures of PA and PGA-based conjugates (DAAV-C). Aim 4. To evaluate the effectiveness of optimized DAAV candidates in various animal models of anthrax infection.